Cancer diagnostics based on plasma protein biomarkers: hard times but great expectations

Abstract

Cancer diagnostics based on the detection of protein biomarkers in blood has promising potential for early detection and continuous monitoring of disease. However, the currently available protein biomarkers and assay formats largely fail to live up to expectations, mainly due to insufficient diagnostic specificity. Here, we discuss what kinds of plasma proteins might prove useful as biomarkers of malignant processes in specific organs. We consider the need to search for biomarkers deep down in the lowest reaches of the proteome, below current detection levels. In this regard, we comment on the poor molecular detection sensitivity of current protein assays compared to nucleic acid detection reactions, and we discuss requirements for achieving detection of vanishingly small amounts of proteins, to ensure detection of early stages of malignant growth through liquid biopsy.

Keywords: affinity-based protein assays; blood markers; liquid biopsy; protein biomarkers; proximity ligation assay; tissue-specific expression.

Fig. 1

Liquid biopsy provides opportunities to diagnose and monitor malignancies anywhere in the body by sampling blood or other body fluids. The figure lists some common targets of liquid biopsy, including relevant categories of proteins.

Fig. 2

Some formats for affinity‐based protein assays. (A) In forward immune assays, immobilized antibodies (light blue) capture target proteins (green) from samples where all proteins have been labeled with detectable moieties (red). (B) In reverse immune assays, samples are immobilized on solid phases for detection by labeled antibodies. (C) Sandwich immune assays employ pairs of antibodies, one immobilized on a solid support to capture target proteins from solution, and one labeled for detection. (D) The Simoa assay is a variant of the sandwich assays where the particle used as a solid support is confined to a compartment so small that even a single bound detection antibody can elicit a detectable signal, well above any nonspecific signals from media. (E) The SomaScan technique uses immobilized, chemically modified DNA strands (dark blue) as affinity reagents. Upon capture of target proteins and washes, the bound proteins are biotinylated (yellow). Next, the DNA strands and any captured and biotinylated proteins are released for renewed capture on another solid support via the added biotin residues. Finally, the secondarily immobilized probe DNA strands are identified and quantified as a measure of detected proteins. (F) In proximity extension assays pairs of antibodies with conjugated, short DNA strands (dark blue) are incubated with sample, followed by dilution of the reaction and enzymatic extension of DNA strands that remain in proximity by virtue of having bound the same target protein molecule. Finally, the extension products are amplified and recorded by real‐time PCR or DNA sequencing. (G) Solid‐phase proximity ligation assays use trios of target‐specific antibodies, one immobilized for target capture, and two labeled with DNA strands. Oligonucleotides on pairs of antibodies brought in proximity by binding the target protein and remaining after washes are joined by enzymatic DNA ligation, giving rise to reporter DNA strands that can be amplified and recorded as a measure of detected target proteins.

Fig. 3

Specific and nonspecific signals in sandwich immune assays. Successful immune reactions give rise to detection signals (red), but contributions to the recorded signal can also arise due to nonspecific signal that may be difficult to distinguish from the correct signals. Such background noise may arise from solutions, reaction vessels, and detection instruments that may exhibit fluorescence or absorbance, etc., that cannot be distinguished from the true signal. Any labeled antibodies that have failed to bind target proteins but that remain after washes due to nonspecific binding will also give rise to background signals. Finally, cross‐reactive binding by the affinity reagents to molecules other than the intended target molecules (yellow) will also contribute to nonspecific signals and thus limit detection signals.